超表达马尾松PmPT3基因提高拟南芥耐低磷能力

doi:10.13560/j.cnki.biotech.bull.1985.2021-0072

摘要/Abstract

摘要：

旨在探讨马尾松PmPT3基因在低磷胁迫条件下对拟南芥磷吸收利用的影响,实现林木优良基因的利用。通过构建马尾松PmPT3基因超表达载体pBWA（V）HS-PmPT3,采用花序浸染法遗传转化拟南芥。经筛选和检测,共获得4株T3代纯合株系;磷处理结果表明:超表达马尾松PmPT3基因显著提高了转基因拟南芥超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）的活性,分别是野生型植株的2.17倍、1.59倍、1.81倍;丙二醛（MDA）含量较野生型植株降低了47.58%。转基因拟南芥的地上部分和根部总磷含量及无机磷含量较野生型相比,分别提高了1.26倍和1.74倍及1.38倍和1.89倍。转基因植株较野生型地上部干重提高了45.46%,根干重提高了55.56%,总干重提高了46.15%。与正常供磷条件相比,处于低磷胁迫下的拟南芥转基因植株,其根部和地上部分中的PmPT3基因表达量显著上调,且在根部达到极显著水平。结果表明,超表达马尾松PmPT3基因可以提高低磷胁迫下拟南芥中保护酶活性、降低丙二醛产生,促进拟南芥对磷元素的吸收,从而提高拟南芥耐低磷胁迫的能力,为通过基因工程创制耐低磷新种质提供了科学依据。

关键词: 超表达, 马尾松, PmPT3基因, 拟南芥, 低磷胁迫

Abstract:

The aim of this study is to explore the effect of PmPT3 gene of Pinus massoniana on phosphorus uptake and utilization in Arabidopsis thaliana under low-phosphorus stress,and to achieve the utilization of excellent genes in trees. The PmPT3 gene overexpression vector pBWA（V）HS-PmPT3 was constructed and transformed into A. thaliana by inflorescence infection method. Four homozygous strains of T3 generation were obtained by screening and detection. The results of phosphorus treatment showed that overexpression of the PmPT3 gene of P. massoniana significantly increased the activities of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）in transgenic Arabidopsis,respectively,it was 2.17 times,1.59 times,and 1.81 times of the wild-type plant. It reduced malondialdehyde（MDA）content,47.58% lower than that of wild-type plants. Compared with the wild-type,the total phosphorus content and inorganic phosphorus content of the aerial parts and roots of transgenic Arabidopsis increased by 1.26 times and 1.74 times,as well as 1.38 times and 1.89 times,respectively. Compared with the wild type,the dry weight of the transgenic plant shoots increased by 45.46%,the dry weight of roots by 55.56%,and the total dry weight by 46.15%.Compared with normal phosphorus supply conditions,the expressions of PmPT3 gene in the root and shoot parts of transgenic A. thaliana under low phosphorus stress was significantly up-regulated,and reached a very significant level in the roots. These results indicated that the overexpression of PmPT3 gene from P. massoniana increased the activities of protective enzymes,reduced the production of MDA,and promoted the absorption of phosphorus in Arabidopsis under low phosphorus stress. Therefore,overexpression of the PmPT3 gene improved the ability of Arabidopsis to tolerate low phosphorus stress. This work may provide a reliable scientific basis for the creation of new low phosphorus tolerant germplasm through genetic engineering.

Key words: overexpression, Pinus massoniana, PmPT3 gene, Arabidopsis thaliana, low phosphorus stress

方丹丹, 张婷, 文晓鹏. 超表达马尾松PmPT3基因提高拟南芥耐低磷能力[J]. 生物技术通报, 2021, 37(10): 1-8.

FANG Dan-dan, ZHANG Ting, WEN Xiao-peng. Overexpression of Pinus massoniana PmPT3 Gene in Arabidopsis thaliana Increasing Low Phosphorus Tolerance[J]. Biotechnology Bulletin, 2021, 37(10): 1-8.

图/表 8

表1 实验所用引物及序列

Table 1 Primers and sequences used in the study

引物Primer	序列Sequence（5'-3'）	退火温度Annealing temperature/℃
PmPT3-F	CAGTGGTCTCACAACATGGGC- GACAATGAGGGG	55
PmPT3-R	CAGTGGTCTCATACACTACAC- GGGCATAGTTCTGT	55
Actin2-qRTF	ACGGTAACATTGTGCTCAGT- GGTG	59
Actin2-qRTR	CTTGGAGATCCACATCTGCTGGA	59
PmPT3-qRTF	CTATCCGCTTTCAGCCACCA	59
PmPT3-qRTR	TTCTCCACACAAAATCCGCC	59

图1 马尾松PmPT3基因植物表达载体的构建 M:DL2000 DNA marker;A:添加酶切位点的PmPT3基因片段;B:BLUNT-PmPT3双酶切产物;C:pBWA（V）HS双酶切产物;D:重组质粒pBWA（V）HS-PmPT3双酶切产物;E:1-11菌落PCR产物

Fig.1 Construction of plant expression vector of P. masso-niana PmPT3 Gene M: DL2000 DNA marker; A: PmPT3 fragment with adding restriction sites; B: BLUNT-PmPT3 enzyme digestion product; C: pBWA(V)HS denzyme digestion product; D: recombinant plasmid pBWA(V) HS-PmPT3 enzyme digestion product; E: 1-11 colony PCR product

图2 马尾松PmPT3遗传转化拟南芥过程 LP:低磷（0.125 mmol/L）;TG:转基因植株;A,B:T1 代转基因拟南芥筛选;C,D:T2 代转基因拟南芥筛选;E,F:T3 代拟南芥纯合株系筛选;G,H:磷处理20 d后拟南芥表型

Fig.2 Process of genetic transformation of P. massoniana PmPT3 into Arabidopsis LP: Low phosphorus (0.125 mmol/L) ; TG; transgenic plants; A, B: T1 generation transgenic Arabidopsis selection; C, D: T2 generation transgenic Arabidopsis selection; E, F: screening of T3 generation pure commonzanara; G, H: phenotype of Arabidopsis thaliana after 20 days of phosphorus treatment

图3 T1代转基因拟南芥qRT-PCR分析（A）及纯合株系PCR检测（B）图中不同小写字母表示在P<0.05水平上差异显著

Fig.3 Analysis of positive gene expression in T1 generation transgenic Arabidopsis thaliana (A) and PCR detection of homozygous strains (B) Different lowercase letters in the figure indicate significant differences at the P<0.05 level

图4 低磷胁迫对转基因拟南芥POD（A）、SOD（B）、CAT（C）活性及MDA（D）含量的影响 LP:低磷（0.125 mmol/L）;TG:转基因植株;图中不同小写字母表示在P<0.05水平上差异显著

Fig.4 Effect of low phosphorus stress on the POD(A), SOD(B), CAT(C) activity and MDA(D) content of transgenic Arabidopsis LP: Low phosphorus (0.125 mmol/L); TG: transgenic plants; different lowercase letters in the figure indicate significant differences at the P<0.05 level. The same below

图5 低磷胁迫对转基因拟南芥总磷含量（A）和无机磷含量（B）的影响

Fig.5 Effect of low phosphorus stress on the total phosphorus content (A) and inorganic phosphorus content (B) of transgenic Arabidopsis

图6 低磷胁迫对转基因拟南芥地上部干重（A）、根干重（B）、总干重（C）和根冠比（D）的影响

Fig.6 Effect of low phosphorus stress on the above-ground dry weight (A), root dry weight (B), total dry weight (C) and root to shoot ratio (D) of transgenic Arabidopsis

图7 磷处理后马尾松PmPT3基因相对表达量

Fig.7 Relative expression of PmPT3 gene of P. massoni-ana after phosphorus treatment

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